Outer layers of plants such as leave cuticle, fruit peels, as well as bark protect the plant against abrasion, prevent water loss, and also protect against pathogenic microorganisms. The breaking through the plant cuticle is a prerequisite for a pathogen to be able to enter the plant's internal tissue.
The mechanism, by which plants naturally defend themselves against this early stage of pathogenesis has not been fully understood. The initial process of fungal propagules attaching to a host plant is essential to the successful establishment of pathogenesis. The established facts that aerial fungal pathogens bind strongly to very hydrophobic surfaces suggests that hydrophobic forces are involved in the attachment processes.
The attachment of aerial fungal pathogens involves an active process of secretion of extracellular mucilages or adhesives, which may start within minutes after contact with the host. Other reported components in the adhesive secretions include enzymes, among them esterases and cutinases. The erosion of cuticular waxes adjacent to and underlying the conidium may became observable within 20 min of liquid release. The growth of the appressorial germ tube appears to be limited to the zone of deposition of the liquid film.
Some studies have suggested that preparation of the infection court involves active dissolution of the host cuticle by foliar pathogen. It is also believed that this dissolution is the purpose of these enzymes. Nicholson, R. L., and L. In: The Fungal Spore and Disease Initiation in Plants and Animals., Eds. Cole, G. T., and Hoch, H. C.,1991 Plenum Press, New York, 3-23. Thus, the cuticle has to be penetrated by the attacking pathogen before the sequential steps of disease development occur. Some fungal spores help themselves with mechanical force exerted by the infection structure in addition to the enzymatic degradation. (Koller, W. in: The Fungal Spore and Disease Initiation in Plants and Animals., Eds. Cole, G. T., and Hoch, H. C., 1991 Plenum Press, New York, 219-246.
Pentacyclic triterpenes (PT) are among the most common plant secondary metabolites, but their function in plants have not been understood. They are usually concentrated in the outermost layers such as plant cuticle, fruit peel and bark. In some cases, these layers contain very high concentration of pentacyclic triterpenes. For example, an apple peel contains about 0.1 grams of ursolic acid per fruit, and the outer bark of white birch species contains up to 40% w/w of betulin. The amount of betulin obtainable from the birch bark waste in the wood-working industry in Finland is estimated at about 150,000 tons per annum. At present, waste bark is used as a low value fuel for energy production. Jaaskelainen, P. (1981) Pap. Puu 63, 599-603.
Literature supplies numerous examples of enzymes that can be inhibited by PT, indicating the ability of PT to act broadly in a non-specific mode on multiple targets. See for example, (a.) Buchler et al. (1991) Biochem. Biophys. Acta 1075, 206-212, Inhibition of rat renal 11.beta.-hydroxysteroid dehydrogenase by steroidal compounds and triterpenoids; structure/function relationship; (b.) Koch et al. (1994) Phytother. Res. 8, 109-111, In vitro inhibition of adenosine deanminase by a group of steroid and triterpenoid compounds.; (c.) Najid et al. (1992) FEBS 299, 213-217, Characterization of ursolic acid as a lipoxygenase and cyclooxygenase inhibitor using macrophages, platelets and differentiated HL60 leukemic cells.; (d.) Pengsuparp et al. (1994) J. Nat. Prod. 57, 415-418, Pentacyclic triterpenes derived from Maprounea africana are potent inhibitors of HIV-1 reverse transcriptase.; (e.) Simon et al. (1992) Biochem. Biophys. Acta 1125, 68-72, Inhibition of lipoxygenase activity and HL60 leukemic cell proliferation by ursolic acid isolated from heather flowers (Calluna vulgaris).; (f.) Ying et al. (1991) Biochem. J. 277, 521-526 Inhibition of human leucocyte elastase by ursolic acid. Evidence for a binding site for pentacyclic triterpenes. The disclosures of each of these references is herein incorporated by reference.
In plant tissue cultures, stress induced by inactivated fungi or fungal enzymes has been used to enhance production of biologically active secondary metabolites. In several instances it has been reported that this fungal elicitation led to overproduction of pentacyclic triterpenes instead of some other expected metabolites. Suitable example is given by Van der Heijden et al., (1988) Plant Cell Rep. 7, 51-54, where tissue cultures of Tabernaemontana spp., normally producing indole alkaloids, were subjected to stress induced by either fungi, bacteria, or enzyme cellulase or pectinase. When stressed, however, the culture produced up to 3.3 times the normal rate of the ursane-type pentacyclic triterpenes (2% of dry mass) but no increase in the production of indole alkaloids occurred.
Other experiments with Tabernaemontana divaricata treated with Candida albicans elicitor led to production of a series of pentacyclic triterpenes of the ursane and oleane types, and was accompanied by inhibition of both growth and indole alkaloid accumulation (Van der Heijden et al., (1989) Phytochemistry 28, 2981-1988).
Also, the tissue culture of Tripterygium wilfordii, normally a source of potent cytotoxic diterpenes, stressed by fungal Botrytis elicitor dramatically enhanced production of oleane-type pentacyclic triterpenes but not the diterpenes, prompting a conclusion that only triterpenes are inducible anti-microbial phytoalexins. Kutney, et al., (1993) Anti-inflammatory oleane triterpenes from Tripterygium wilfordii cell suspension cultures by fungal elicitation. Plant Cell Rep. 12, 356-359.
The conventional treatments for leafy and grassy plants that have been attacked by fungi and bacteria are usually exercised after an outbreak occurs. The affected plants are then treated with one or more of the commercial synthetic contact antimicrobial sprays at application rates that do not pose phytotoxicity concerns. Effective treatment rates must be balanced against the risks of harming the treated plant with chemicals that are structurally unrelated to those in the plant physiology. It would be desirable to have a protective agent that is effective against microbial pathogens which would also work in a manner analogous to the plant's natural defense mechanisms to reduce the risk of phytotoxicity. One of the problems associated with treating hydrophobic leaf surfaces is an effective application of the material. Current spraying techniques result in a portion of the sprayed material falling to the ground directly or after being washed off from rain occurring shortly after application. Either event increases concerns for environmental contamination.
Protective agents that are applied by spraying should remain on the plant surface for a time sufficient to serve their intended function. Stability against ultraviolet and visible light is, therefore, a concern for foliar treatments. It would be desirable to develop fungicides for foliar application that resist degradation by exposure to ultraviolet as well as visible light.
Moreover, invading organisms have been known to evade the effects of a treatment agent by mutation and propagation of resistant strains. Such developed resistance is economically detrimental because it forces the discovery of new treatments. It would be helpful to provide plant anti-infective agents which cannot be evaded by mutating pathogens.